HIV vaccines employing the Envelope glycoprotein (Env) as an immunogen have not met their initial promise, in part, because this approach has failed to induce a broadly neutralizing antibody (bnAb) response. Our key innovation is the generation and use of homogeneous glycopeptide immunogens to initiate and focus an immune response against the HIV envelope glycoprotein. These glycopeptide immunogens will form the basis of an HIV vaccine. HIV evades neutralization by displaying many epitopes that result in a dominant non-neutralizing response in humans. However, over the last ten years, dozens of broadly neutralizing antibodies and their cognate epitopes have been discovered and rigorously characterized, thus providing a roadmap to develop potential vaccines. The glycopeptides and candidate immunogens produced by Chemitope leverage these discoveries and faithfully mimic the essential structural features of these broadly neutralizing epitopes by adopting the targeted structure and by displaying a defined glycosylation pattern. Therefore, our immunogens 1) focus the immune response by binding with high affinity to low-frequency unmutated common ancestor (UCA) precursor B cells, 2) guide these B cells toward mature breadth and potency and 3) avoid building a non-productive immune response to the distracting, non-neutralizing epitopes. Previously, we have synthesized several candidate immunogens derived from the first and second variable region of Env, V1V2. Our most recent lead pre-clinical candidate V1V2 GPd displays excellent antigenicity toward the inferred UCA and mature bnAbs PG9 and CH01. Unfortunately, the antigenic conformation of this glycopeptide is unstable in vivo. Solving this conformational instability is the focus of this proposal. In this project, we will develop candidate immunogen glycopeptides that will be chemically stable and retain antigenicity throughout vaccine formulation. Folding conditions will be developed to draw each glycopeptide into the desired stable conformation, locked through covalent bond-formation. Feasibility will be evaluated for our most promising glycopeptide constructs in CH01 UCA knock-in mice to determine if they are viable vaccine candidates. Aim I: Synthesize a series of linear V1V2 Glycopeptide Immunogens. Aim II: Chemically lock the V1V2 Glycopeptide Immunogens in the antigenic conformation. Aim III: Initiate the expansion and maturation of CH01 UCA B cells in a transgenic mouse model.